STROMELYSIN-1 DEFICIENT TRANSGENIC MICE

Abstract: A transgenic mouse with alterations in a stromelysin-1 gene is prepared by introduction of a gene encoding an altered stromelysin-1 gene into a host mouse.

Description: TITLE OF THE INVENTION STROMELYSIN-1 DEFICIENT TRANSGENIC MICE FIELD OF THE INVENTION The present invention relates to transgenic nonhuman animals wherein a stromelysin- 1 gene is altered. CROSS-REFERENCE TO RELATED APPLICATION This is a continuation-in-part of U.S. Serial No. 08/034,929, filed March 19, 1993, now pending. BACKGROUND OF THE INVENTION Stromelysin, collagenase and gelatinase are matrix metalloendoproteinases secreted by fϊbroblasts and chondrocytes. The enzymes are capable of degrading the major connective tissue components of articular cartilage or basement membranes. Elevated levels of both enzymes have been detected in joints of arthritic humans and animals (K. A. Hasty et al., "The role of stromelysin in the cartilage destruction that accompanies inflammatory arthritis", Arthr. Rheum. 33; 388-97 (1990); S. M. Krane et al., "Modulation of matrix synthesis and degradation in joint inflammation" in The Control of Tissue Damage, A. B. Glauert (ed.), Elsevier Sci. Publ., Amsterdam, (1988,) pp 179-95; A. Blanckaert et al-, "Direct extraction and assay of collagenase from human osteoarthritic cartilage", Clin. Chim. Acta, 185: 73-80 (1989)). Each enzyme is secreted from these cells as an inactive proenzyme which is subsequently activated. There is evidence that stromelysin may be the in vivo activator for collagenase, implying a cascade for degradative enzyme activity (A. Ho et al., "Evidence that human rheumatoid synovial matrix metalloproteinase 3 is an endogenous activator of procollagenase", Arch. Biochem. Biophys., 267: 211-16 (1988); G. Murphy et al., "Stromelysin is an activator of procollagenase", Biochem. J., 248: 265-8 (1987); Y. Ogata et al., "Matrix metalprotease-3 (stromelysin) activates the precusor for human matrix metaloproteinase-9", J. Biol. Chem. 267: 3581-84 (1992)). Inhibition of stromelysin may limit the activation of collagenase and gelatinase as well as prevent the direct degradation of extracellular matrix proteins by stromelysin. That inhibition of stromelysin may be effective in preventing articular cartilage, degradation has been demonstrated in vitro by measuring the effect of matrix metalloendoproteinase inhibitors on proteoglycan release from rabbit cartilage explants (C. B. Caputo et al., J. Orthopaedic Res., 6: 103-8 (1988)). There is an extensive literature on the involvement of these metalloproteinases in diseases such as arthritis, but there is very little to guide one in developing a specific inhibitor for each enzyme. Human rheumatoid synovial collagenase has been shown to share ~ 50% homology with human stromelysin (S. E. Whitham et al., "Comparison of human stromelysin and collagenase by cloning and sequence analysis", Biochem. J., 240: 913-6 (1986)). Gelatinase (MR ~ 72,000) has been isolated from rheumatoid fibroblasts (Y. Okada et al., "Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts", Eur. J. Biochem., 194: 721-30 (1990)). The synthesis of the proenzyme is not coordinately regulated with stromelysin and collagenase, and the activation of gelatinase may also be different. The role of gelatinase in the destruction of articular cartilage appears to be different from the other enzymes; therefore, inhibition of gelatinase may protect tissues from degradation. A higher molecular weight gelatinase (MR ~ 95,000) is also secreted by fibroblasts and monocytes and may be involved in cartilage degradation. Inhibitors of stromelysin and collagenase are believed to have utility in preventing articular cartilage damage associated with septic arthritis. Bacterial infections of the joints can elicit an inflammatory response that may be perpetuated beyond that which is needed for removal of the infective agent. The inflammation may result in permanent damage to structural components. Bacterial components have been used in animal models to elicit an arthritic response with the appearance of proteolytic activities (J. P. Case et al-, "Transin/-stromelysin expression in the synovium of rats with experimental erosive arthritis", J. Clin Invest, 84, 1731-40 (1989); R. J. Williams et ah, "Septic Arthritis: Staphylococcal induction of chondrocyte proteolytic activity", Arthr. Rheum., 33: 533-41 (1990)). Inhibitors of stromelysin, collagenase, and gelatinase are believed to be useful to control tumor metastasis, optionally in combination with current chemotherapy and/or radiation (L. M. Matrisian et ah, "The mRNA coding for the secreted protease transin is expressed more abundantly in malignant than in benign tumors", Proc. Natl. Acad. Sci. USA, 83: 9413-7 (1986); S. M. Wilhelm, et al., "Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells", Proc. Natl. Aca. Sci, USA, 84: 6725-29 (1987); Z. Werb et al.," Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression", J. Cell Biol., 109: 872-889 (1989); L. A. Liotta et ah, "Tumor invasion and the extracellular matrix", Lab. Invest., 49: 636-649 (1983); R. Reich et al., "Inhibitors of collagenase IV and cell adhesion reduce the invasive activity of malignant tumor cells", in Metastasis: Ciba Foundation Symposium; Wiley, Chi Chester, 1988, pp. 193-210.). Secreted proteinases such as stromelysin, collagenase, and gelatinase play an important role in processes involved in the movement of cells during metastasic tumor invasion. There is also evidence that the matrix metalloproteinases are overexpressed in certain metastatic tumor cell lines. In this context, the enzyme penetrates underlying basement membranes, thus allowing the tumor cell to escape from the site of primary tumor formation and enter circulation. After adhering to blood vessel walls, the tumor cells use these metalloendoproteinases to pierce underlying basement membranes and penetrate other tissues, thereby leading to tumor metastasis. Inhibition of this process would slow metastasis and improve the efficacy of chemotherapeutics or radiation treatments. Inhibitors of matrix metalloproteinases should also be useful for controlling periodontal diseases, such as gingivitis. Both collagenase and stromelysin activities have been isolated from fibroblasts isolated from inflamed gingiva (V. J. Uitto et ah, "Collagenase and neutral metalloproteinase activity in extracts of inflamed human gingiva", J. Periodontal Res., 16: 417-424 (1981 )). Enzyme levels have been correlated to the severity of gum disease (C. M. Overall et al., "Demonstration of tissue collagenase activity in vivo and its relationship to inflammation severity in human gingiva", J. Periodontal Res., 22: 81-88 (1987)). Proteolytic processes have also been observed in the ulceration of the cornea following alkali burns (S. I. Brown et al., "Collagenolytic activity of alkali-burned corneas", Arch. Opthalmol., 81 : 370-373 (1969)). Mercapto -containing peptides inhibit collagenase isolated from alkali-burned rabbit cornea (F. R. Burns et al., Invest. Opthalmol., 30: 1569-1575 (1989)). Treatment of alkali- burned eyes or corneal ulcers resulting from bacterial infection with metalloendo-proteinase inhibitors in combination with sodium citrate or sodium ascorbate or antimicrobials may prevent corneal degradation. Stromelysin has been implicated in the degradation of structural components of the glomerular basement membrane (GBM) of the kidney, which restricts passage of plasma proteins into the urine (W. H. Baricos et aL, "Degradation of glomerular basement membrane by purified mammalian metalloproteinases", Biochem. J., 254: 609-612 (1988)). Proteinuria, a result of glomerular disease, is excess protein in the urine caused by increased permeability of the GBM to plasma proteins. Inhibition of stromelysin may alleviate the proteinura associated with kidney malfunction. Inhibition of stromelysin activity may prevent rupturing of atherosclerotic plaques. The tearing or rupture of atherosclerotic plaques is the most common event initiating coronary thrombosis. Destabilization and degradation of the connective tissue matrix surrounding these plaques by proteolytic enzymes or cytokines released by infiltrating inflammatory cells has been proposed as a cause of plaque fissuring. Such tearing of these plaques can cause an acute thrombolytic event as blood rapidly flows out of the blood vessel. High levels of stromelysin mRNA have been found to be localized to individual cells in atherosclerotic plaques removed from heart transplant patients at the time of surgery (A. M. Henney et al., "Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization", Proc. Nat'l. Acad. Sci. USA, 88: 8154-8158 (1991)). Inhibition of stromelysin activity may prevent or delay the degradation of the connective tissue matrix that stabilizes the atherosclerotic plaques, thereby preventing or delaying events leading to acute coronary thrombosis. It is also believed that inhibitors of matrix metallo¬ proteinases would have utility in treating degenerative aortic disease associated with thinning of the medial aortic wall. Aneurysms are often associated with atherosclerosis in this tissue. Increased levels of the degradative activities of the matrix metalloproteinases have been identified in patients with aortic aneurysms and aortic stenosis (N. Vine et aL, "Metalloproteinases in degenerative aortic diseases", Clin, Sci., 81 : 233-9 (1991)). Inhibition of these enzymes may aid in preventing or delaying the degradation of aortic tissue, thus preventing events leading to acute and often times fatal aortic aneurysms. It is also believed that specific inhibitors of stromelysin and collagenase should be useful as birth control agents. There is evidence that expression of metalloendoproteinases, including stromelysin and collagenase, is observed in unfertilized eggs and zygotes and at further cleavage stages and increased at the blastocyst stage of fetal development and with endoderm differentiation (C. A. Brenner et aL_ "Genes for extracellular matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development", Genes & Develop., 3: 848-59 (1989)). A blastocyst may express metallo-proteinases in order to penetrate the extracellular matrix of the uterine wall during implantation. Inhibition of stromelysin and collagenase during these early developmental processes should presumably prevent normal embryonic development or implantation in the uterus. Such intervention would constitute a novel method of birth control. In addition there is evidence that collagenase is important in ovulation processes. In this example, a covering of collagen over the apical region of the follicle must be penetrated in order for the ovum to escape. Collagenase has been detected during this process and an inhibitor of collagenase has been shown to be effective in preventing ovulation (J. F. Woessner et al., "Connective tissue breakdown in ovulation", Steroids, 54: 491-499 (1989)). There may also be a role for stromelysin activity during ovulation (C. K. L. Too et al., "Relaxin increases the release of plasminogen activator, collagenase, and proteo-glycanase from rat granulosa cells in vitro". Endocrin., 1 15: 1043-1050 (1984)). Collagenolytic and stromelysin activity have also been observed in dystrophobic epidermolysis bullosa (A. Kronberger et al., J. Invest. Dermatol., 79 208-211 (1982); D. Sawamura et ak, Biochem. Biophys. Res. Commun., 174: 1003-8 (1991)). Inhibition of metalloendoproteinases should limit the rapid destruction of connective components of the skin. The precise roles of stromelysin in normal tissue development and maintenance, as well as in embryonal and fetal development, are not known at this time. Due to the putative biological importance of stromelysin in arthritis, as well as in tumor metastasis, the stromelysin- 1 (SLN-1) gene is an important target for embryonic stem (ES) cell manipulation. The generation of SLN-1 deficient transgenic mice would aid in defining the normal role(s) of stromelysin, and allow an animal model of SLN-1 deficiency to be used in the design and assessment of chemical approaches to inhibiting stromelysin activity. Such SLN-1 deficient transgenic mice can also be used as a source of cells for cell culture. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a sequence comparison of exons 2, 3, and 4 derived from cosmid clone cES-11 (top sequence) with a partial murine transin- 1 (stromelysin- 1) cDNA pTRl l (bottom sequence) (L. Ostrowski et al., Mol. Carcinogenesis 1 :13-19 (1988)). The exons from cES-11 were 100% identical to the sequence of the murine stromelysin-1 cDNA clone, confirming that cES-11 contains the mouse SLN-1 gene. Figure 2 is a sequence comparison of exons 2, 3 and 5 derived from cosmid clone cES-4 (top sequence) with a partial murine stromelysin- 1 cDNA pTRl l (bottom sequence) (T. Bowden, University of Arizona). The exons from cES-4 were 89% identical to the sequence of the murine stromelysin- 1 cDNA clone, indicating that cES-4 does not contain the mouse SLN-1 gene. cES-4 exons were most identical (92%) to rat stromelysin-2 (transin-2) suggesting that cES-4 contains the murine stromelysin-2 gene. Figure 3 is a comparison of stromelysin (transin) catalytic domain amino acid sequences. The amino acid sequences of the catalytic domains (Muller et aL, Biochem. J. 253: 187-192) were compared the isolated murine SLN-1 and partial SLN-2 genes of the present invention to determine their homology to human SLN-1 and rat SLN-1. Figure 4 is a genomic map of the mouse stromelysin- 1 gene contained on murine genomic clone cES-11. Single letter codes are Ac, AccI; Ap, Apal; B, BamHI; E, EcoRI; Ev, EcoRV; H, Hindlll; Hn, HincJJ; K, Kpnl; Nh, Nhel; P, PstI; and Xb, Xbal. The exons of mouse SLN-1 are shown as black boxes; exons 1 - 7 proceed 5' to 3' as shown on the map. The genomic clone cES-11 ends after exon 7, and the missing 3' end of SLN-1 is indicated by the dashed line. Mouse repetitive elements are indicated by the encircled R's, with the size of the symbol indicating the apparent relative abundance of the repeat domain. Figure 5 is the predicted disruption of the mouse chromosomal stromelysin- 1 gene by targeted gene replacement using replacement vector pSl -RVl.l. Single letter codes are Ac, AccI; Ap, Apal; B, BamHI; E, EcoRI; Ev, EcoRV; H, Hindlll; Hn, Hincϋ; K, Kpnl; Nh, Nhel; P, PstI; and Xb, Xbal. The exons of mouse SLN-1 are shown as black boxes; exons 1 - 7 proceed 5' to 3' as shown on the map. The genomic clone ends after exon 7, and the missing 3' end of SLN-1 is indicated by the dashed line. Mouse repetitive elements are indicated by the encircled R's, with the size of the symbol indicating the apparent relative abundance of the repeat domain. Recombination with pRVl .l was designed to insert the neo gene into the SLN-1 gene and remove most of SLN-1 exon 3 and all of exon 4, which contain a large portion of the murine SLN-1 catalytic domain. Targeted homologous recombination with the replacement vector pSl-RVl.l is indicated by the large crosses. The arrows indicate the location and direction of PCR primers to be used for identification of pRVl .l targeted cell lines. The black bars over the map indicate the location of unique regions isolated as probes for Southern hybridization confirmation of the targeted alleles. The PCR control vector p5'Slneo which was used to mimic SLN-1 targeted recombination is shown. Figure 6 is a Southern hybridization analysis of targeted embryonic stem (ES) clones. Bottom) Stromelysin exons 1 - 7 are depicted by black boxes, and EcoRI sites are indicated by the letter 'E'. Digestion of with EcoRI followed by hybridization with the 5' flanking probe (gray box) yields a 20 kb wild-type SLN-1 fragment a 4.7 kb targeted SLN-1 allele. Top) Southern analysis of ES cell genomic DNA yielded a 20 kb wild-type SLN-1 fragment. Three SLN-1 targeted (PCR positive) ES cell lines yielded two hybridizing fragments, one wild-type 20 kb fragment and one targeted 4.7 kb fragment. Figure 7 is a Southern hybridization analysis of heterozygous (FI) transgenic mice. Genomic DNA from two transgenic (TG) heterozygous mice yielded one wild-type (w.t.) SLN- 1 fragment and one targeted (KO) fragment for both EcoRI and BamHI digests. Genomic DNA from the nontransgenic (NT) littermate only contained the wild-type fragments. This pattern was confirmed using both 5' and 3' unique probes. Figure 8 is a Southern hybridization analysis of homozygous (F2) transgenic mice. Southern analysis of genomic DNA from F2 (het. x het. crosses) yielded the following patterns. 1) wild-type SLN-1+/+ mice containing only the 20 kb wild-type SLN-1 fragments, 2) homozygous SLN-1-/- mice containing two targeted 4.7 kb fragments, and 3 and 4) heterozygous SLN-1+/- mice containing one wild-type 20 kb fragment and one targeted 4.7 kb fragment. Figure 9 is a Northern hybridization analysis of stromelysin- 1 inactivation in the knock-out mice. Wild-type (+/+) or knock-out (-/-) 14 day mouse embryos were used to prepare primary mouse embryo fibroblasts. Cultures were stimulated with murine IL-1 (1 nM a and β) ('IL-1 * ) or unstimulated ('U') for 48 hours, total RNA was isolated, and Northern hybridizations were performed using a murine stromelysin- 1 specific cDNA fragment (Nhel to Hindi, exons 8/9). As expected, the knock-out primary embryonic fibroblasts did not exhibit any detectable stromelysin- 1 expression. Figure 10 is a Western hybridization analysis of stromelysin- 1 inactivation in the knock-out mice. Wild-type (+/+) or knock-out (-/-) 14 day mouse embryos were used to prepare primary mouse embryo fibroblasts. Cultures were unstimulated with murine IL-1 (1 nM a, β or both) in serum-free medium, medium was removed at 2, 4, and 6 days, and Western hybridizations were performed using a sheep anti-rabbit stromelysin antibody. As expected, the knock-out primary embryonic fibroblasts did not exhibit any detectable stromelysin- 1 expression. DETAILED DESCRIPTION OF THE INVENTION The term "animal" is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A "transgenic animal" is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus. The term "transgenic animal" is not intended to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule. This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that alteration or information, then they, too, are transgenic animals. The alteration or information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene. The altered stromelysin- 1 gene generally should not fully encode the same stromelysin- 1 as native to the host animal, and its expression product should be altered to a minor or great degree, or absent altogether. However, it is conceivable that a more modestly modified stromelysin gene will fall within the compass of the present invention if it is a specific alteration. The genes used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof. A type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells may be obtained from pre- implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al., Nature 292: 154-156 (1981); M. O. Bradley et al., Nature 309: 255-258 (1984); Gossler et al- Proc. Natl. Acad. Sci. USA 83: 9065-9069 (1986); and Robertson et al-, Nature 322, 445- 448 (1986)). Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus -mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Science 240: 1468-1474 (1988)). Since matrix metalloproteinase is an independent component of a complex mechanism, the enzymes, including that encoded by SLN-1 , must be examined both individually and as a group if their contribution to the mechanisms of arthritis, metastasis, development and fertility are understood. One approach to the problem of determining the contributions of individual genes and their expression products is to use isolated metalloproteinase genes to selectively inactivate the native wild-type gene in totipotent ES cells (such as those described above) and then generate transgenic mice. The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described 1987 (Thomas et al., Cell 51 :503-512, (1987)) and is reviewed elsewhere (Frohman et al., Cell 56:145-147 (1989); Capecchi, Trends in Genet. 5:70-76 (1989); Baribault et ah, Mol. Biol. Med. 6:481-492, (1989); Wagner, EMBO J. 9: 3025-3032 (1990); Bradley et al., Bio/Technology 10: 534-539 (1992)). Techniques are available to inactivate or alter any genetic region to any mutation desired by using targeted homologous recombination to insert specific changes into chromosomal alleles. However, in comparison with homologous extrachromosomal recombination, which occurs at frequencies approaching 100% homologous plasmid-chromosome recombination was originally reported to only be detected at frequencies between 10-6 and 10-3 (Lin et al., Proc. Natl. Acad. Sci. USA 82:1391 -1395 (1985); Smithies et al, Nature 317: 230-234 (1985); Thomas et al., Cell 44:419-428, (1986); Song et al, Proc. Natl. Acad. Sci. USA 84:6820-6824 (1987)). Nonhomologous plasmid-chromosome interactions are more frequent, occurring at levels 105-fold (Lin et ak, Proc. Natl. Acad. Sci. USA 82:1391-1395 (1985)) to 102-fold (Thomas et ah, Cell 44:419-428 (1986); Song et al, Proc. Natl. Acad. Sci. USA 84:6820-6824 (1987)) greater than comparable homologous insertion. To overcome this low proportion of targeted recombination in murine ES cells, various strategies have been developed to detect or select rare homologous recombinants. One approach for detecting homologous alteration events uses the polymerase chain reaction (PCR) to screen pools of transformant cells for homologous insertion, followed by screening individual clones (Kim et al, Nucleic Acids Res. 16:8887-8903 (1988); Kim et al, Gene 103:227-233 (1991)). Alternatively, a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly (Sedivy et al, Proc. Natl. Acad. Sci. USA 86:227-231 (1989)). One of the most powerful approaches developed for selecting homologous recombinants is the positive-negative selection (PNS) method developed for genes (such as SLN-1) for which no direct selection of the alteration exists (Mansour et al., Nature 336:348-352: (1988); Capecchi, Science 244:1288-1292, (1989); Capecchi, Trends in Genet. 5:70-76 (1989)). The PNS method is more efficient for targeting genes which are not expressed at high levels because the marker gene has its own promoter. Nonhomologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with the herpes drags such as gancyclovir (GANC) or FIAU (l-(2-deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil). By this counter-selection, the number of homologous recombinants in the surviving transformants can be increased. As used herein, a "targeted gene" or "Knock-out" (KO) is a DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the above described methods. The targeted genes of the invention include DNA sequences which are designed to specifically alter cognate endogenouos alleles. The following is presented by way of examples and is not to be construed as a limitation on the scope of the invention. EXAMPLE 1 Preparation of murine embryonic stem cell genomic libraries Evidence suggested that homologous genomic targeting in embryonic stem (ES) cells is strongly inhibited (>100x) by subtle base-pair differences in their genomic DNAs (Riele et al, Proc. Natl. Acad. Sci. USA 89:5128-5132 (1992); Deng et al, Mol. Cell. Biol. 12:3365-3371 (1992)). To circumvent this potential problem, genomic libraries were constructed from ES cells grown in the absence of feeder cell layers for the isolation of genes to be used for subsequent ES cell targeting. Genomic libraries were prepared from ES-J1 or ES-D3 cells according to the in situ procedure described in (Mudgett et al, Genomics 8:623-633, (1990)). The cosmid vector sCos-1 was chosen, as it allows both the vector and the insert to be dephosphorylated. This prevents concantamer formation and generally results in genomic libraries of better quality and quantity (up to 5x106 clones per package) than is achieved with other vectors (Evans et al, Gene 79:9-20, (1989)). Three genomic libraries were constructed with different bacterial hosts to allow for a more representative and stable cloning of methylated and repetitive genomic sequences. Escherichia coli HB 101 host cells maintained sequence direct repeats more stably and more effectively than other bacterial strains. E. coli NM554 host strain was used to allow more effective isolation of methylated genomic elements. The SURE host line (Stratagene) was used to stably maintain indirect repeats and allow isolation of methylated DNA. EXAMPLE 2 Isolation of mouse stromelysin cosmid clones Three ES cell libraries of Example 1 were screened using a partial mouse stromelysin- 1 cDNA (pTRl 1 ) which contained stromelysin exon 2 through most of exon 10 (Ostrowski et al., supra). The conditions for in situ localization of plasmid DNA in R. coli colonies are described by Sambrook et al in "Molecular cloning: a laboratory manual" Cold Spring Harbor Press, (1989). The hybridization conditions were maintained at high stringency to reduce the chances of isolating different metalloproteinases and related pseudogenes. Primary clones were purified through secondary and tertiary screenings. DNAs for the positive cosmids were prepared using standard conditions (Sambrook et al, supra). Two of the screened genomic libraries yielded nine independent stromelysin clones each; a third library screening yielded four cosmid clones. Cosmid DNAs were digested with different restriction endonucleases, electrophoresed on agarose gels, and hybridized with the partial pTRll cDNA insert using standard conditions (Sambrook £!ϋl, supra). Southern hybridization analyses indicated that three of the cosmid clones (one from each library) were from one hybridizing genomic locus (subsequently identified as stromelysin- 1), and that at least sixteen clones were from a distinct hybridizing genomic locus (subsequently identified as stromelysin-2). EXAMPLE 3 Characterization of the mouse stromelysin clones The cosmid clones for mouse stromelysin- 1 and mouse stromelysin-2 of Example 2 were mapped with restriction endonucleases by end-ordered partial digestion (Evans et al, Gene 79:9-20, (1989)). The location and extent of the SLN hybridizing regions of both stromelysin genes were localized by hybridizing complete and end-ordered partial digestions of the cosmids with the mouse pTRll cDNA probe. The restriction maps for both mouse stromelysin genes were compared. No apparent regions of overlap between the mouse loci were detected. To further investigate the possibility of overlapping loci, cosmids cES-11 (SLN-1) and cES-4 (SLN-2) were digested with EcoRI, Hindiπ and PstI; and the resulting cosmid fragments of similar size were isolated. None of these fragments hybridized to the other cosmid clone (SLN locus), suggesting that the two isolated mouse stromelysin loci were distinct and non-overlapping. The two isolated mouse stromelysin genes (designated as SLN-1 and SLN-2) were characterized by nucleotide sequencing of exon regions to confirm their identities as mouse SLN-1 and SLN-2 using standard conditions (Sambrook et al., supra.) Cosmid clones cES-11, cES-22, and cES-31 contained exon nucleic acid sequences which were 100% identical to those contained in the partial murine SLN-1 cDNA pTRl 1 (Fig. 1), and therefore contained the murine stromelysin- 1 gene. Cosmids which appeared to contain the SLN-2 gene (e.g., cES-4), were found to contain exons with specific sequence nucleotide differences from the SLN-1 gene (89%; Fig. 2). The predicted amino acid sequences of the SLN- 1 and SLN-2 catalytic domains were compared to those of human and rat SLN-1 and SLN-2 proteins. It was determined that mouse SLN-1 was most similar to human SLN-1 (Fig. 3); human SLN-1 is associated with rheumatoid arthritis and osteoarthritis. Mouse SLN- 1 was also very similar to rat SLN-1 (Fig. 3); expression of rat SLN- 1 has been found to be elevated in rat models of arthritis (Case et al, J. Clinical Invest. 84:1731 -1740; Hasty et al., Arthritis Rheum. 33:388-397). Due to these similarities, the mouse SLN-1 gene was chosen as the target for gene inactivation studies in mouse embryonic stem (ES) cells and for the generation of stromelysin- 1 deficient transgenic mice. The mouse SLN-1 gene (cosmids cES-1 1 , cES-22, and cES-31) was characterized to determine restriction endonuclease sites, exon locations, and mouse repetitive element domains (Fig. 4). Mouse stromelysin- 1 orientation and exon locations were determined by DNA sequencing, digestion with restriction endonuclease and Southern hybridization analysis (Sambrook et al., supra). It was determined that cES-1 1 contains the 5' promoter region and exons one through seven of mouse SLN-1. The genomic material of cES-11 terminated in the intron between exons seven and eight, and did not contain exons eight through ten. The 7.5 kb EcoRI restriction fragment which contained the mouse SLN- 1 gene was subcloned into pSKII (Stratagene) for further analysis and construction of SLN-1 targeting vectors. The location of mouse repetitive DNA in the mouse stromelysinal genomic clone was determined by using total mouse genomic DNA as a hybridization probe. Hybridization kinetics allowed only the abundant DNA repetitive classes to give hybridization signals; therefore, this approach effectively defined areas of high and moderate mouse DNA repeats (Fig. 4). The locations of these elements were important in the constraction of targeting vectors and genomic probes as the genomic probes must be derived from nonrepetitive DNA. EXAMPLE 4 Constraction of stromelysin- 1 gene targeting and PCR control vectors From the knowledge of the genomic organization of mouse SLN-1 gene with regard to restriction sites, exons, and repetitive domains (Example 3), gene replacement vectors for inactivating the stromelysin gene were prepared using standard cloning techniques (Sambrook et al., supra). The gene replacement vector design chosen for the first gene replacement, vector pSl- RV1.1 , contained a 3.5 kb PstI stromelysin- 1 gene fragment as the long arm, a selectable neo marker inserted into the Hindlll site of exon three (in the catalytic domain), and a HSV-TK marker gene attached to the 1.2 kb PstI to Hindlll stromelysin- 1 gene fragment short arm (Fig. 5). The neo marker was inserted to express in the antisense direction of the SLN-1 target gene. Selection against the HSV-TK gene with FIAU allowed for the enhancement of targeted recombinants as described (Mansour et al, Nature 336:348-352, (1988); Capecchi, Science 244:1288-1292, (1989); Capecchi, Trends in Genet. 5:70-76 (1989)). As the neo and HSV-TK genes are both driven by the highly efficient mouse phosphoglycerate kinase-1 (PGK-1) promoter, these marker genes (a gift from R. Jaenisch, The Whitehead Institute) were inserted into the targeting vector as indirect repeats to create a more stable vector. Use of pSl-RVl .l in gene replacement resulted in the insertion of the neo marker in the SLN-1 catalytic region and the deletion of most of the SLN-1 sequences which encode the catalytic domain (Fig. 5). The total length of SLN homology in pS l-RVl.l is 4.7 kb. Primers and control vectors were constructed and used in PCR assays to detect the inactivation of the SLN-1 gene with pS l- RV1.1. One primer originated from a region 5' of the pSl-RVl .l short arm in the mouse SLN-1 promoter (5'- GAGGAGCCAGAGAACCTAC TGAAGG-3'; SEQ. ID: 1), and the second primer originated from the 3' end of the neo marker gene (5'- TCATAGCCTGAAGAACGAGA TCAGC-3'; SEQ. ID: 2) (Fig. 5). Only SLN-1 genes having a neo marker inserted in the correct location yielded the correct PCR product. To act as a control for the PCR identification of targeted ES cell lines, a vector (p5'Slneo) was constructed which contained sequences 5' of the SLN-1 short arm, the short arm itself, and the correctly placed neo marker (Fig. 5). This arrangement precisely mimicked the predicted outcome of a targeted recombination which inactivates a genomic SLN-1 allele. p5'Slneo was transfected and randomly inserted into ES cells to control for the geneticin selection of the neo marker insertion; this allowed ES clones with random p5'S lneo insertions to be used as positive controls for the PCR- dependent identification of SLN-1 targeting with pSl-RVl .l . Genomic DNA isolated from these transformants was used to optimize the PCR reaction for identification of ES cell clones which contain a targeted SLN-1 gene. The optimal PCR conditions for use of these primers in detecting the targeted SLN-1 genes were 50 mM Tris, pH 8.5, 50 mM NaCl, 1.5 mM MgCl2, and 2 mM DTT, and 1.25 U Taq enzyme. The PCR reaction was run at 94°C for 30 sec, 62°C for 45 sec, and 72°C for 3 min, using 40 cycles of reaction. EXAMPLE 5 Targeted alteration of the stromelysin- 1 gene in murine ES cells The gene replacement vector used in the SLN-1 gene disraption experiments was the pRVl .l vector of Example 4. When this vector recombined with the wild-type SLN-1 allele to generate the SLN-1 knock-out (SLN-1 KO), exons 3 and 4 of the catalytic domain were deleted (Fig. 5). The mouse embryonic stem cell line AB2.1 was electroporated with Pvul-linearized pSl -RVl . l in multiple experiments. All AB2.1 ES cells were cultured on SNL feeder cells as described (Robertson, in Teratocarcinomas and embryonic stem cells, IRL Press, pp. 71-112 (1987)). Electroporations were performed with 4x10 ES cells and 25 μg linearized pRVl .l in 0.8 ml electroporation buffer (Specialty Media, Inc.) at 300v, 250 μF using a Bio-Rad Gene Pulser. ES cell transformants were selected with the antibiotic geneticin (Gibco G418: 200 μg/ml active G418), and some transformants were counter-selected with FIAU (Bristol Myers Squibb; 0.2 μM) for enhancement of homologous recombinants. Murine leukemia inhibitory factor (LIF; ESGRO, Gibco BRL, Inc.) was used at 250- 500 U/ml. Selection with FIAU resulted in about sixfold fewer background pRVl .l transformants, thereby enhancing the isolation of targeted transformants. G418- and FIAU-resistant ES clones were isolated by picking and growing individual colonies, and their genomic DNAs were prepared for AB2.1 RV 1.1 -transformants as described (Laird et al., Nucleic Acids Res. 19:4293 (1991). A clone pooling approach improved the chances of isolating targeted ES cell lines. Thirty pools of about 15 FIAU- enhanced pRVl .l -transformed AB2.1 clones each were isolated, genomic DNA samples were prepared (Laird et al-, supra), and five pools were shown to be positive for SLN-1 targeting by PCR analysis (Example 4). By this pooling approach, the targeting efficiency of SLN-1 with pRVl.l appeared to be about 1/150 FIAU resistant clones. Cells from four of the positive pools were plated on feeder cells, single ES clones were isolated from each pool, and their genomic DNAs were analyzed by PCR analysis. Individual PCR- positive clones were isolated from each of these PCR-positive pools. The frequency of positive single clones isolated from the positive pools (of about 15 clones each) was 15/227, or about 1/15 as expected. Jl knock-out lines were generated using the replacement vector pRVl .l in pooling experiments as previously described for the AB2.1 cell line. Twenty-four pools of about 15 FIAU-enhanced Jl transformants were assayed by PCR analysis; two positive pools were identified. Single clones from the PCR-positive pools were isolated and assayed by PCR analysis, allowing us to identify single PCR-positive Jl clones which contained a targeted SLN-1 allele. To confirm the targeted disraption of the mouse SLN- 1 allele in these PCR-positive single ES cell lines, their genomic DNAs were digested with restriction endonucleases, transferred to Bio-Rad ZetaProbe GT membranes, and genomic Southern hybridizations were performed according to manufacturers instructions. The ES cell genomic DNAs were hybridized with the neo gene, as well as 5' and 3' flanking unique sequence DNA probes. The 5' unique probe resides (5'-3') from the EcoRI site to the Ball site, and the 3' unique probe resides (5'-3') from the Ball site to the EcoRI site (Fig. 5). Digestion and hybridization of genomic DNA from a SLN-1 targeted ES cell line yielded two hybridizing fragments, one wild-type 20 kb (allele) and one targeted 4.7 kb allele (Fig. 6). When the PCR- positive ES cell lines were analyzed in this manner, all cell lines contained both hybridizing fragments. Further Southern hybridization analysis using all three hybridization probes and different restriction endonucleases confirmed that these clones contain the targeted (knocked-out) SLN-1 allele, and that the regions flanking the SLN-1 target were unaltered. EXAMPLE 6 Injection of SLN-1 KO clones into donor blastocysts All SLN- 1 -targeted AB2.1 cell lines were characterized by PCR and Southern hybridization analysis to confirm that SLN-1 was disrupted. The cell lines were grown in culture and characterized. Targeted cell lines which grew normally and did not contain an abnormal proportion of differentiated cells (Robertson, in supra) were then separated from their feeder cells by treating the cell culture with trypsin, allowing the feeder cell to attach for 30-45 min, and removing the unattached ES cells. The ES cells were injected into recipient blastocysts. Three SLN-1 targeted ES clones (a329, a594, and a627) were injected into C57B1/6J recipient blastocysts in separate experiments using techniques described previously (Bradley, A. "Production and analysis of chimeric mice. In Teratocarcinomas and Embryonic Stem Cells: A Practical Approach", E.J. Robertson, ed.Oxford:IRL Press, (1987), ppl 13- 151). The injected C57B1/6J recipient blastocysts were reimplanted into the uterii of day 3 pseudopregnant Tac:SW(fBR) mice and allowed to develop to term. Progeny were screened initially by coat color chimerism, the agouti color (which is the ES cell background strain) being an indicator of ES cell chimerism. SLN-1 targeting was confirmed by PCR and Southern hybridization analysis performed on genomic DNA isolated from tail samples obtained from these mice. Injection of the SLN-1 targeted line a627 yielded 6 progeny mice, of which three were male coat color chimerics. As the ES cell line AB2.1 is homozygous for the agouti (A) coat color gene, penetrance of ES cells into the injected (black coat color) C57B1/6 blastocyst gives rise to chimeric coat color mice. EXAMPLE 7 Breeding chimeric mice The three chimeric coat color male mice from the first injection were bred to wild-type C57B1/6 (black coated) and 129/J (agouti coated) female mice. Some of the progeny from the chimera X C57B1/6 cross were expected to be agouti if the chimeric male had ES cell genetic material incorporated into its germline (agouti is dominant to black coat color). The chimera X 129/J cross would yield only agouti mice. These crosses were performed to transfer a627 ES cell genetic information, including the disrupted SLN-1 allele, to its offspring. To determine the SLN-1 genotypes, genomic DNA was purified from about 1 cm of tail from each mouse after weaning. The genomic DNA was isolated as described (Laird et al, supra), followed by phenol and phenolxhloroform extractions and ethanol precipitation. PCR and Southern hybridization analysis (as described in Example 5) were used to identify offspring which contained the disrupted SLN-1 allele. These transgenic offspring were heterozygous for the SLN-1 disraption. Both transgenic heterozygous and nontransgenic mouse (tail) genomic DNAs were digested with both EcoRI and BamHI, and were hybridized with 5' and 3' flanking DNA probes to confirm the transgenic SLN-1 structure (Fig. 7). Southern hybridization analysis confirmed that the structure of the altered stromelysin allele was identical to that predicted, and previously characterized in the SLN-1 targeted ES clones. EXAMPLE 8 Breeding heterozygous mice and generation of homozygous stromelysin- 1 deficient mice Male and female transgenic mice, each of which contained one copy of the altered stromelysin- 1 allele (heterozygous mice), were mated with each other to generate mice in which both copies of the stromelysin- 1 gene were the targeted, altered transgenic SLN-1 gene. It was predicted that one fourth of the mouse embryos would be homozygous for the altered stromelysin- 1 gene. Surviving offspring were genotyped by Southern hybridization as described above (Fig. 8). It was determined that 21 % of the 147 offspring mice were homozygous SLN-1 -/-, 22 (24%) were wild-type SLN- 1+/+, and 52% were heterozygous SLN-1 -/+. These numbers indicate that there was no significant decrease in the number of stromelysin- 1 deficient transgenic mice which survived past weaning; however the number of females was less than predicted. EXAMPLE 9 Characterization of homozygous stromelysin- 1 deficient mice Surviving homozygous stromelysin- 1 deficient mice of Example 8 were bred with wild-type or heterozygous mates to determine if they were fertile. All homozygous SLN-1 -/- males and females tested were fertile. Significant differences in gross morphology or histology between the stromelysin- 1 deficient mice and the wild-type or heterozygous mice were not observed. EXAMPLE 10 Confirmation of stromelysin- 1 inactivation by Northern and Western analysis Wild-type (SLN-1+/+) or know-out (SLN-1-/-) 14 day mouse embryos were dissected to remove the heads and internal organs, and the tissue from 6-1" embryos (each genotype) was minced and trypsinized for 30 minutes at 37°C. Cells were washed in media, grown for 24 hours in DMEM/10% fetal bovine serum (FBS), and were replated at 3x10^ cells per 75 cm2 flask. After 24 hours, the cells were washed with serum-free medium, and were grown in serum-free DMEM for Western and Northern analysis of DMEM/10% FBS for Northern analysis. Wild-type and knock-out embryonic fibroblast cultures were cultured in the absence of interleukin-1 (IL-1) (uninduced), or in IL-lα, IL-lβ, or both IL-lα and IL-lβ at a concentration of 1 nM. After 48 hours, cells were washed and total RNA was isolated by standard procedures. Agarose gel electrophoresis and Northern hybridizations were carried out by standard procedures using a stromelysin- 1 cDNA fragment (Nhel to Hindi) specific for stromelysin- 1 hybridization. After 2, 4, and 6 days, 1 ml aliquots of medium were concentrated to 1.10 volume, and polyacrylamide gel electrophoresis and Western hybridizations were carried out by standard procedures using a sheep anti-rabbit stromelysin antibody. As expected, the knock-out primary embryonic fibroblasts did not exhibit any detectable stromelysin- 1 expression by both Northern and Western analysis. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Mudgett, John S Hutchinson, Nancy (ii) TITLE OF INVENTION: Stromelysin-1 Deficient Transgenic Mice (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Christine E. Carty (B) STREET: P.O. Box 2000 RY60-30 (C) CITY: Rahway (D) STATE: New Jersey (E) COUNTRY: USA (F) ZIP: 07065 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MΞ-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US 08/034,929 (B) FILING DATE: 19-MAR-1993 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Carty, Christine E (B) REGISTRATION NUMBER: 36,099 (C) REFERENCE/DOCKET NUMBER: 18969IAP (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (908) 594-6734 (B) TELEFAX: (908) 594-4720 (C) TELEX: 138825 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : GAGGAGCCAG AGAACCTACT GAAGG 25 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : TCATAGCCTG AAGAACGAGA TCAGC 25 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 612 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : AGAAAACTAC TATGGCCTTG CAAAAGATGT GAAGCAATTT ATTAAGAAAA AGGACAGTAG 60 TCTTATTGTC AAAAAAATTC AAGAAATGCA GAAGTTCCTC GGGTTGGAGA TGACAGGGAA 120 GCTGGACTCC AACACTATGG AGCTGATGCA TAAGCCCAGG TGTGGTGTTC CTGATGTTGG 180 TGGCTTCAGT ACCTTCCCAG GTTCGCCAAA ATGGAGGAAA TCCCACATCA CCTACAGGTA 240 ATGGCACCAG TTACCTTCTT TGATTTCTCT TATCCAGGAT TGTGAATTAT ACACCGGATT 300 TGCCAAGACA GAGTGTGGAT TCTGCCATTG AAAAAGCTTT GAAGGTCTGG GAGGAGGTGA 360 CCCCACTCAC TTTCTCCAGG ATCTCTGAAG GAGAGGCTGA CATAATGATC TCCTTTGCAG 420 TTGGAGGTAA AAGCCAAAAA CTACCTGAAA ATAACATATG TATGTATGTA TTTTCTTTTT 480 AAGGAACATG GAGACTTTGT CCCTTTTGAT GGGCCTGGAA CAGTCTTGGC TCATGCCTAT 540 GCACCTGGAC CAGGGATTAA TGGAGATGCT CACTTTGACG ATGATGAACG ATGGACAGAG 600 GATGTCACTG GT 612 (2) INFORMATION FOR SEQ ID NO:4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 551 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AGAAAAATAC TACAACTTTA CAAAAAATGA GACCAAATTT TCCAAAAGGA ACGACAGTAG 60 TCCTGTTGTC AAAAAAATTC AAGAAATGCA GAAGTTCCTC GGGTTGGAGA TGACAGGGAA 120 GCTGGACTCC AACACTATGG AGCTGATGCA CAAGCCCAGG TGTGGTGTTC CTGATGTTGG 180 TGGCTTCAGT ACCTTCCCAG GTTCGCCAAA ATGGAGGAAA TCCCACATCA CCTACAGGAT 240 TGTGAATTAT ACACCAGATT TGCCAAGACA GAGTGTGGAT TCTGCCATTG AGAAAGCTTT 300 GAAGGTCTGG GAGGAGGTGA CCCCACTCAC TTTCTCCAGG ATCTCTGAAG GAGAGGCTGA 360 CATAATGAAC TCCTTTGCAG TTGGAGGACC AACTTATTCC TGGTTGCAGC CCATGAACTT 420 GGCCACTCCC TGGGTCTCTT TCATTCCGAC AAGAAAGAAT CTCTGATGTA CCCAGTCTAC 480 AGGTTCTCCA CAAGCCCAGC TAACTTCCAC CTTTCTCAAG ATGATATAGA GGGCATTCAA 540 TCCCTGTATG G 551

Claims: WHAT IS CLAIMED IS: 1. A transgenic animal whose somatic and germ cells contain a gene coding for an altered form of stromelysin- 1, the altered gene having been targeted to replace wild-type stromelysin- 1 allele into the animal or an ancestor of the animal at an embryonic stage using embryonic stem cells, the altered gene containing specific deletions of stromelysin exon DNA and stromelysin introm DNA, and the altered gene containing a neo gene. The transgenic animal of Claim 1 which is a mouse. 3. The mouse of Claim 2, the mouse being fertile and capable of transmitting the altered stromelysin- 1 gene to its offspring. 4. The mouse of Claim 2 wherein the altered stromelysin- 1 gene has been introduced into an ancestor of the mouse at an embryonic stage by microinjection of altered embryonic stem cells into mouse blastocyts. 5. The mouse of Claim 2 wherein the altered stromelysin- 1 gene has been introduced into the mouse at an embryonic stage by microinjection of altered embryonic stem cells into mouse blastocyts. 6. The mouse of Claim 2 which is designated SLN-1. 7. A method of producing a mouse of Claim 2 which compπses: (a) providing a gene encoding an altered form of stromelysin- 1 designed to target a stromelysin- 1 allele of mouse embryonic stem cells; (b) introducing the altered gene into mouse embryonic stem cells; (c) selecting embryonic stem cells which contain the altered gene; (d) injecting the embryonic stem cells containing the altered stromelysin- 1 gene into mouse blastocysts; (e) transplanting the injected blastocysts into a pseudopregnant mouse, and (f) allowing the embryo to develop to term; to produce a founder transgenic mouse. 8. The method of Claim 7 wherein the introduction is by microinjection. 9. The method of Claim 7 producing a mouse of Claim 2 which further comprises: (g) breeding the chimeric transgenic mice to wild- type mice to obtain heterozygous (FI) mice; and (h) breeding the heterozygous (FI) mice to generate homozygous (F2) stromelysin- 1 deficient transgenic mice. 10. A cell line derived from the transgenic animal of Claim 1.

Inventors: HUTCHINSON NANCY, MUDGETT JOHN S, HUTCHINSON, NANCY, MUDGETT, JOHN, S.
Applicants: HUTCHINSON NANCY, MERCK & CO INC, MUDGETT JOHN S, HUTCHINSON, NANCY, MERCK & CO., INC., MUDGETT, JOHN, S.
Application year: 1994
Main classification: C12N5
IPC codes: A01K67, C12N5, C12N9, C12N15, C12R1